Severe neurologic manifestations from cervical spine instability in spondylo-megaepiphyseal-metaphyseal dysplasia

Spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD; OMIM 613330) is a dysostosis/dysplasia caused by recessive mutations in the homeobox-containing gene, NKX3-2 (formerly known as BAPX1). Because of the rarity of the condition, its diagnostic features and natural course are not well known. We describe clinical and radiographic findings in six patients (five of which with homozygous mutations in the NKX3-2 gene) and highlight the unusual and severe changes in the cervical spine and the neurologic complications. In individuals with SMMD, the trunk and the neck are short, while the limbs, fingers and toes are disproportionately long. Radiographs show a severe ossification delay of the vertebral bodies with sagittal and coronal clefts, missing ossification of the pubic bones, large round "balloon-like" epiphyses of the long bones, and presence of multiple pseudoepiphyses at all metacarpals and phalanges. Reduced or absent ossification of the cervical vertebrae leads to cervical instability with anterior or posterior kinking of the cervical spine (swan neck-like deformity, kyknodysostosis). As a result of the cervical spine instability or deformation, five of six patients in our series suffered cervical cord injury that manifested clinically as limb spasticity. Although the number of individuals observed is small, the high incidence of cervical spine deformation in SMMD is unique among skeletal dysplasias. Early diagnosis of SMMD by recognition of the radiographic pattern might prevent of the neurologic complications via prophylactic cervical spine stabilization.     

一种用于CYP3A5基因分型的电化学传感器阵列设计

目的：设计一种用于检测CYP3A5基因分型的电化学传感器阵列及其不同基因型的判别方法。方法：设计的电化学基体由印刷电路板（PCB）组成,该电路板包含一组金电极。每个金电极表面修饰有包含单链捕获探针的自组装单分子膜。设计中使用二茂铁做为电活性指示剂,基因分型检测是通过两种不同电势的二茂铁衍生物分别标记等位基因特异性信号探针来实现。结果：该设计能构建一种快速准确、操作简便的DNA电化学传感器阵列检测系统。结论：本文设计为使用电化学方法检测基因分型提供了一种新方法和新技术。     

Profiling of cytokine expression by biotin-labeled-based protein arrays

Global analysis of protein expression holds great promise in basic research and patient care. Previously we demonstrated that multiple cytokines could be detected simultaneously using an enzyme-linked immunosorbent assay protein array system with high sensitivity and specificity. In this paper, we described a biotin-labeled-based protein array system to detect multiple cytokines simultaneously from biological samples. In this new approach, proteins from a variety of biological sources are labeled with biotin. The biotin-labeled proteins are then incubated with antibody chips. Targeted proteins are captured by the array antibodies spotted on the antibody chips. The presence of targeted proteins is detected using Cy3- or Cy5-conjugated streptavidin and signals are imaged by laser scanner. The system also can be easily adapted to a two-color binding assay, allowing measurement of the levels of proteins in a test sample with respect to a reference sample at the same chip. To demonstrate its potential applications, we applied this technology to profile human cytokines, chemokines, growth factors, angiogenic factors and proteases in estrogen receptor (ER)+ and ER- cells. These results suggest that biotin-labeled-based antibody chip technology can provide a practical and powerful means of profiling hundreds or thousands of proteins for research and clinical purposes.     

Microbioreactor arrays with parametric control for high-throughput experimentation

A scalable array technology for parametric control of high-throughput cell cultivations is demonstrated. The technology makes use of commercial printed circuit board (PCB) technology, integrated circuit sensors, and an electrochemical gas generation system. We present results for an array of eight 250 microl microbioreactors. Each bioreactor contains an independently addressable suite that provides closed-loop temperature control, generates feed gas electrochemically, and continuously monitors optical density. The PCB technology allows for the assembly of additional off-the-shelf components into the microbioreactor array; we demonstrate the use of a commercial ISFET chip to continuously monitor culture pH. The electrochemical dosing system provides a powerful paradigm for reproducible gas delivery to high-density arrays of microreactors. Growth data are presented for Escherichia coli cultured in the array with varying microaerobic conditions using electrochemically generated oxygen. Additionally, we present data on carbon dioxide generation for pH dosing.     

一种用于CYP3A5基因分型的电化学传感器阵列设计

目的:设计一种用于检测CYP3A5基因分型的电化学传感器阵列及其不同基因型的判别方法。方法:设计的电化学基体由印刷电路板(PCB)组成,该电路板包含一组金电极。每个金电极表面修饰有包含单链捕获探针的自组装单分子膜。设计中使用二茂铁做为电活性指示剂,基因分型检测是通过两种不同电势的二茂铁衍生物分别标记等位基因特异性信号探针来实现。结果:该设计能构建一种快速准确、操作简便的DNA电化学传感器阵列检测系统。结论:本文设计为使用电化学方法检测基因分型提供了一种新方法和新技术。     

Fabrication of DNA microarrays onto polymer substrates using UV modification protocols with integration into microfluidic platforms for the sensing of low-abundant DNA point mutations

We describe the microfabrication and operational characteristics of a simple flow-through biochip sensor capable of detecting low abundant point mutations in K-ras oncogenes from genomic DNA, which carry high diagnostic value for colorectal cancers. The biochip consisted of an allele-specific ligase detection reaction (LDR) coupled to a universal array for interrogating multiple mutations simultaneously from a clinical sample. The integrated sensing platform was micro-manufactured from two different polymers, polycarbonate, PC, which was used for the LDRs, and poly(methyl methacrylate), PMMA, which was used to build the microarray. Passive elements were hot embossed into the PC and PMMA microchips and then, the chips assembled into a three-dimensional architecture with the interconnect fabricated from an elastomer, poly(dimethylsiloxane), PDMS, to produce a leak-free connection between the biochips. The array in PMMA was produced using a photomodification process, which involved three steps; (1) UV (254 nm) exposure of the polymer surface; (2) EDC coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond) and; (3) washing of the surface. The LDR/hybridization flow-through biochip performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time=19.1 min) and could screen multiple mutations simultaneously for high throughput applications at a level of one mutant sequence in 100 wild-type sequences.     

Microarray genotyping resource to determine population stratification in genetic association studies of complex disease

We have developed a robust microarray genotyping chip that will help advance studies in genetic epidemiology. In population-based genetic association studies of complex disease, there could be hidden genetic substructure in the study populations, resulting in false-positive associations. Such population stratification may confound efforts to identify true associations between genotype/haplotype and phenotype. Methods relying on genotyping additional null single nucleotide polymorphism (SNP) markers have been proposed, such as genomic control (GC) and structured association (SA), to correct association tests for population stratification. If there is an association of a disease with null SNPs, this suggests that there is a population subset with different genetic background plus different disease susceptibility. Genotyping over 100 null SNPs in the large numbers of patient and control DNA samples that are required in genetic association studies can be prohibitively expensive. We have therefore developed and tested a resequencing chip based on arrayed primer extension (APEX) from over 2000 DNA probe features that facilitate multiple interrogations of each SNP, providing a powerful, accurate, and economical means to simultaneously determine the genotypes at 110 null SNP loci in any individual. Based on 1141 known genotypes from other research groups, our GC SNP chip has an accuracy of 98.5%, including non-calls.     

Characterization of multicomponent monosaccharide solutions using an enzyme-based sensor array

We report the development of a sensor for rapidly and simultaneously measuring multiple sugars in aqueous samples. In this strategy, enzyme-based assays are localized within an array of individually addressable sites on a micromachined silicon chip. Microspheres derivatized with monosaccharide-specific dehydrogenases are distributed to pyramidal cavities anisotropically etched in a wafer of silicon (100) and are exposed to sample solution that is forced through the cavities by a liquid chromatography pumping system. Production of fluorescent reporter molecules is monitored under stopped-flow conditions when localized dehydrogenase enzyme systems are exposed to their target sugars. We demonstrate the capability of this analysis strategy to quantify beta-D-glucose and beta-D-galactose at low micromolar to millimolar levels, with no detectable cross-talk between assay sites. Analysis is achieved either through fluorescence detection of an initial dehydrogenase product (NADH, NADPH) or by production of a secondary fluorescent product created by hydride transfer from the reduced nicotinamide cofactor to a fluorogenic reagent. The array format of this sensor provides capabilities for redundant analysis of sugars and for monitoring levels of other solution components known to affect the activity of enzymes. The use of this strategy to normalize raw fluorescence signals is demonstrated by the determination of glucose and pH on a single chip. Alternatively, uncertainties in the activity of an immobilized enzyme can be accounted for using standard additions, an approach used here in the determination of serum glucose.     

Development of a High-Throughput Resequencing Array for the Detection of Pathogenic Mutations in Osteogenesis Imperfecta

Objective

Osteogenesis imperfecta (OI) is a rare inherited skeletal disease, characterized by bone fragility and low bone density. The mutations in this disorder have been widely reported to be on various exonal hotspots of the candidate genes, including COL1A1, COL1A2, CRTAP, LEPRE1, and FKBP10, thus creating a great demand for precise genetic tests. However, large genome sizes make the process daunting and the analyses, inefficient and expensive. Therefore, we aimed at developing a fast, accurate, efficient, and cheaper sequencing platform for OI diagnosis; and to this end, use of an advanced array-based technique was proposed.

Method

A CustomSeq Affymetrix Resequencing Array was established for high-throughput sequencing of five genes simultaneously. Genomic DNA extraction from 13 OI patients and 85 normal controls and amplification using long-range PCR (LR-PCR) were followed by DNA fragmentation and chip hybridization, according to standard Affymetrix protocols. Hybridization signals were determined using GeneChip Sequence Analysis Software (GSEQ). To examine the feasibility, the outcome from new resequencing approach was validated by conventional capillary sequencing method.

Result

Overall call rates using resequencing array was 96–98% and the agreement between microarray and capillary sequencing was 99.99%. 11 out of 13 OI patients with pathogenic mutations were successfully detected by the chip analysis without adjustment, and one mutation could also be identified using manual visual inspection.

Conclusion

A high-throughput resequencing array was developed that detects the disease-associated mutations in OI, providing a potential tool to facilitate large-scale genetic screening for OI patients. Through this method, a novel mutation was also found.     

Tag/anti-tag liquid-phase primer extension array: a flexible and versatile genotyping platform

This study demonstrates an array-based platform to genotype simultaneously single nucleotide polymorphisms (SNPs) and some short insertions/deletions (indels) by the integration of the universal tag/anti-tag (TAT) system, liquid-phase primer extension (LIPEX), and a novel two-color detection strategy on an array format (TATLIPEXA). The TAT system permits a universal chip to be used for many applications, and the LIPEX simplifies the sample preparation but improves the sensitivity significantly. More importantly, all SNPs and some short indels can be interrogated in a single reaction with only two fluorescent ddNTPs. The concept of TATLIPEXA is demonstrated for nine SNPs (eight point mutations and one single-base insertion), and genotypes obtained show a remarkable concordance rate of 100% with both DNA sequencing and restriction fragment length polymorphism. Moreover, TATLIPEXA is able to provide quantitative information on allele frequency in pooled DNA samples, which could serve as a rapid screening tool for SNPs associated with diseases.     

DNA suspension arrays: silencing discrete artifacts for high-sensitivity applications

Detection of low frequency single nucleotide polymorphisms (SNPs) has important implications in early screening for tumorgenesis, genetic disorders and pathogen drug resistance. Nucleic acid arrays are a powerful tool for genome-scale SNP analysis, but detection of low-frequency SNPs in a mixed population on an array is problematic. We demonstrate a model assay for HIV-1 drug resistance mutations, wherein ligase discrimination products are collected on a suspension array. In developing this system, we discovered that signal from multiple polymorphisms was obscured by two discrete hybridization artifacts. Specifically: 1) tethering of unligated probes on the template DNA elicited false signal and 2) unpredictable probe secondary structures impaired probe capture and suppressed legitimate signal from the array. Two sets of oligonucleotides were used to disrupt these structures; one to displace unligated reporter labels from the bead-bound species and another to occupy sequences which interfered with array hybridization. This artifact silencing system resulted in a mean 21-fold increased sensitivity for 29 minority variants of 17 codons in our model assay for mutations most commonly associated with HIV-1 drug resistance. Furthermore, since the artifacts we characterized are not unique to our system, their specific inhibition might improve the quality of data from solid-state microarrays as well as from the growing number of multiple analyte suspension arrays relying on sequence-specific nucleic acid target capture.     

Electrochemical detection of nucleic base mismatches with ferrocenyl naphthalene diimide

Electrochemical detection of nucleic base mismatches was attempted successfully with ferrocenyl naphthalene diimide (FND) in a model system with 20-meric double-stranded oligonucleotides with or without a mismatch(es). Thus, dA(20) or a 20-meric sequence of the lac Z gene was immobilized on a gold electrode and complementary oligonucleotides with different numbers of mismatches were allowed to hybridize in the presence of FND to give rise to an electrochemical signal. The signal intensity varied depending on the number of unpaired bases on the DNA duplex. From experiments with a quartz crystal microbalance, eight molecules of FND were found to bind to the 20-meric double-stranded oligos and this number decreased as the number of mismatches increased. These findings were further supported by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. This novel method will be useful for the analysis of single-nucleotide polymorphisms present on human genes.     

Immunoassays based on microelectrodes arrayed on a silicon chip for high throughput screening of liver fibrosis markers in human serum

A novel immunoassays for screening of disease markers in human serum are presented by miniaturizing interdigitated array (IDA) of microelectrodes via micro electro-mechanical system (MEMS) on a silicon chip for multi-channel electrochemical measurement. Different selected antibodies (Abs) are incorporated site-specifically into the electrochemically deposited polypyrrole (PPy) formed on the IDA of the silicon chip, which was characterized by fluorescence microscope photo and the electrochemical quartz crystal microbalance (EQCM) measurements. The selective recognition of Ab to the corresponding antigen (Ag) is monitored through the measurable conductivity change, which is directly visualized by cyclic voltammograms (CVs) in presence of the redox probe, Fe (CN)₆^3−/4−. By using the strategy presented here, three liver fibrosis markers, hyaluronic acid (HA), lamin (LN) and collagen type IV (IV-C), are detected simultaneously and specifically at the surface of the chip with calibration curves, y = 21.75 + 0.84x (R = 0.995), y = 57.54 + 0.47x (R = 0.999) and y = 37.92 + 0.28x (R = 0.999), separately. Either the standard or the serum samples can be detected at ng/mL concentration level in a tiny amount of volume, 50 μL. The chip-based immunoassay shows the advantages of high sensitivity, good specificity, high throughput, low sample consumption, and the stability offered via batch production by MEMS as well, which is expected to benefit the multi-target screening of desired clinical analytes.     

Grating-coupled surface plasmon resonance: a cell and protein microarray platform

Grating-coupled surface plasmon resonance (GCSPR) is a method for the accurate assessment of analyte in a multiplexed format using small amounts of sample. In GCSPR, the analyte is flowed across specific receptors (e.g. antibodies or other proteins) that have been immobilized on a sensor chip. The chip surface is illuminated with p-polarized light that couples to the gold surface's electrons to form a surface plasmon. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs, thus reducing the intensity of reflected light. Shifts in the GCSPR angle can be correlated with refractive index increases following analyte capture by chip-bound receptors. Because regions of the chip can be independently analyzed, this system can assess 400 interactions between analyte and receptor on a single chip. We have used this label-free system to assess a number of molecules of immunological interest. GCSPR can simultaneously detect an array of cytokines and other proteins using the same chip. Moreover, GCSPR is also compatible with assessments of antigen expression by intact cells, detecting cellular apoptosis and identifying T cells and B cells. This technology represents a powerful new approach to the analysis of cells and molecular constituents of biological samples.     

Multianalyte immunoassay based on insulating-controllable PoPD film at arrayed electrodes integrated on a silicon chip

A novel, simple and label-free multianalyte immunoassay system is presented here by integrating arrayed electrodes on a silicon chip via MEMS. The chip is consisted of six Au disk electrodes, an Au counter electrode and an Ag/AgCl reference electrode. Semi-insulating poly(o-phenylenediamine) (PoPD) was utilized to co-polymerize and immobilize antibodies at the arrayed Au electrodes, and wider linear detection range was obtained than those prepared with completely insulating PoPD. Electrochemical cyclic voltammogram (CV), AC impedance spectroscopy, AFM and fluorescence microscopy were employed to characterize the system. The arrayed electrodes offered exact control of deposition position via electrochemical operation, allowing selectively immobilization of different antibodies at desired positions on a single chip. Specific recognition of antibody (Ab) to corresponding antigen (An) was quantitatively monitored by cyclic voltammograms in the presence of electrochemical redox probe, ferrocene methanol. The proposed immunoassay chips showed sensitive response to three liver fibrosis markers, hyaluronic acid (HA), collagen type IV (IV-C) and lamin (LN) at ng/mL level simultaneously and specifically in a tiny amount of volume, usually 50 μL. The results obtained via chips were well consistent with those obtained by commercial radio immunoassays (RIA).     

Identification and biosynthesis of cyclic enterobacterial common antigen in Escherichia coli

Phosphoglyceride-linked enterobacterial common antigen (ECA(PG)) is a cell surface glycolipid that is synthesized by all gram-negative enteric bacteria. The carbohydrate portion of ECA(PG) consists of linear heteropolysaccharide chains comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. The potential reducing terminal GlcNAc residue of each polysaccharide chain is linked via phosphodiester linkage to a phosphoglyceride aglycone. We demonstrate here the occurrence of a water-soluble cyclic form of enterobacterial common antigen, ECA(CYC), purified from Escherichia coli strains B and K-12 with solution nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and additional biochemical methods. The ECA(CYC) molecules lacked an aglycone and contained four trisaccharide repeat units that were nonstoichiometrically substituted with up to four O-acetyl groups. ECA(CYC) was not detected in mutant strains that possessed null mutations in the wecA, wecF, and wecG genes of the wec gene cluster. These observations corroborate the structural data obtained by NMR and ESI-MS analyses and show for the first time that the trisaccharide repeat units of ECA(CYC) and ECA(PG) are assembled by a common biosynthetic pathway.     

Simultaneous intra- and extracellular superoxide monitoring using an integrated optical and electrochemical sensor system

A new integrated optical and electrochemical sensor system for simultaneous monitoring of intra- and extracellular superoxide (O(2)(-)) was developed using an array-based cell chip. For in vitro assays, A172 human glioblastoma cells were transferred into the cell chip and stimulated by phorbol 12-myristate 13-acetate (PMA). Intracellular O(2)(-) generation was detected via fluorescence image analysis with a dye probe, dihydrorhodamine 123 (DHR 123). Extracellular O(2)(-) was detected using an amperometric sensor constructed by immobilisation of cytochrome c using a binder, 3,3'-dithiobis(sulphosuccinimidylpropionate), to attach the redox protein onto the surface of electrodeposited Au electrodes incorporated into the optically transparent cell chip. The simultaneous intra- and extracellular production of O(2)(-) was successfully observed from PMA-stimulated A172 cells and inhibited by superoxide dismutase (SOD). The quantification of O(2)(-) concentration based on a mathematical model study and possible applications using the sensor system developed were discussed. The results confirm that there was no detectable interference or crosstalk between the optical and electrochemical assays. Feasibility of the integration of the two methods, optical and electrochemical, and the neutralisation of the intra- and extracellular O(2)(-) levels by SOD have been demonstrated.     

Genetic risk factors for insidious equine recurrent uveitis in Appaloosa horses

Appaloosa horses are predisposed to equine recurrent uveitis (ERU), an immune‐mediated disease characterized by recurring inflammation of the uveal tract in the eye, which is the leading cause of blindness in horses. Nine genetic markers from the ECA1 region responsible for the spotted coat color of Appaloosa horses, and 13 microsatellites spanning the equine major histocompatibility complex (ELA) on ECA20, were evaluated for association with ERU in a group of 53 Appaloosa ERU cases and 43 healthy Appaloosa controls. Three markers were significantly associated (corrected P‐value <0.05): a SNP within intron 11 of the TRPM1 gene on ECA1, an ELA class I microsatellite located near the boundary of the ELA class III and class II regions and an ELA class II microsatellite located in intron 1 of the DRA gene. Association between these three genetic markers and the ERU phenotype was confirmed in a second population of 24 insidious ERU Appaloosa cases and 16 Appaloosa controls. The relative odds of being an ERU case for each allele of these three markers were estimated by fitting a logistic mixed model with each of the associated markers independently and with all three markers simultaneously. The risk model using these markers classified ~80% of ERU cases and 75% of controls in the second population as moderate or high risk, and low risk respectively. Future studies to refine the associations at ECA1 and ELA loci and identify functional variants could uncover alleles conferring susceptibility to ERU in Appaloosa horses.     

Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology

The technology and application of arrayed primer extension (APEX) is presented. We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imaging, and data analysis. The method is based upon an array of oligonucleotides, immobilized via the 5' end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a change in the color code of the primer sites. The technology was applied to the analysis of 10 common beta-thalassemia mutations. Nine patient DNA samples, each of which carries a different mutation, and four wild-type DNA samples were correctly identified. The signal-to-noise ratio of this technology is, on the average, 40:1, which enables the identification of heterozygous mutations with a high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms.     

A Genome-Wide Association Study Identifies Risk Loci to Equine Recurrent Uveitis in German Warmblood Horses

Equine recurrent uveitis (ERU) is a common eye disease affecting up to 3–15% of the horse population. A genome-wide association study (GWAS) using the Illumina equine SNP50 bead chip was performed to identify loci conferring risk to ERU. The sample included a total of 144 German warmblood horses. A GWAS showed a significant single nucleotide polymorphism (SNP) on horse chromosome (ECA) 20 at 49.3 Mb, with IL-17A and IL-17F being the closest genes. This locus explained a fraction of 23% of the phenotypic variance for ERU. A GWAS taking into account the severity of ERU, revealed a SNP on ECA18 nearby to the crystalline gene cluster CRYGA-CRYGF. For both genomic regions on ECA18 and 20, significantly associated haplotypes containing the genome-wide significant SNPs could be demonstrated. In conclusion, our results are indicative for a genetic component regulating the possible critical role of IL-17A and IL-17F in the pathogenesis of ERU. The associated SNP on ECA18 may be indicative for cataract formation in the course of ERU.